The allergic immune response is initiated by crosslinking of immunoglobulin E (IgE) antibodies bound to receptors (Fc-epsilon-RI) on the surface of mast cells, stimulating a signaling cascade that results in exocytosis of secretory granules (degranulation) to release chemical mediators of allergy and inflammation. IgE crosslinking on mast cells also stimulates exocytosis of recycling endosomes (REs) in a process that, like degranulation, depends on calcium mobilization. The long term goal of this research is to determine the roles of RE exocytosis in mast cell function. To this end: we will define protein components required for antigen-stimulated RE exocytosis; we will develop a imaging method to visualize this process directly; and we will characterize the relationship of this process to other events activated by antigen-mediated IgE crosslinking, including phagocytosis and cytokine secretion. A key objective in this work is to identify proteins important for stimulated RE exocytosis in mast cells, and likely candidates emergent from previous studies will be examined, including small GTPases and SNARE proteins. Both siRNA-mediated knockdown and dominant negative protein expression will be used to assess the roles of specific proteins in RE exocytosis in a novel assay that follows the outward trafficking of green fluorescent protein-labeled glycosylphosphatidylinositol-anchored protein (GFP-GPI). Subsequent studies will use genetic inhibition of RE exocytosis to examine its roles in antigen-mediated phagocytosis and cytokine secretion. In addition, a photoactivatable form of GFP-GPI will be constructed and used to visualize stimulated RE membrane trafficking and fusion in individual cells. Microfabricated arrays of ligands will be used to characterize the spatial relationship between RE exocytosis and activation via IgE-Fc-epsilon-RI that are concentrated in defined patterns. The proposed studies will advance our understanding of the functional roles of recycling endosomes in mast cells and, in general, of stimulated membrane trafficking in cell biology and immunology.